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n e f r o l o g i a. 2 0 1 5;3 5(5):421–447

Revista de la Sociedad Española de Nefrología

w w w.rev is tanef ro logia .com

eview

n update for atypical haemolytic uraemic syndrome:iagnosis and treatment. A consensus document�

osep M. Campistola,∗, Manuel Ariasb, Gema Aricetac, Miguel Blascoa, Laura Espinosad,ario Espinosae, Josep M. Grinyóf, Manuel Macíag, Santiago Mendizábalh,anuel Pragai, Elena Románh, Roser Torra j, Francisco Valdésk, Ramón Vilaltac,

antiago Rodríguez de Córdobal

Servicio de Nefrología, Hospital Clínic, Barcelona, SpainServicio de Nefrología, Hospital Universitario Marqués de Valdecilla, Santander, SpainServicio de Nefrología Pediátrica, Hospital Universitari Materno-Infantil Vall d’Hebrón, Universidad Autónoma de Barcelona, Barcelona,painServicio de Nefrología Pediátrica, Hospital La Paz, Madrid, SpainServicio de Nefrología, Hospital Universitario Reina Sofía, Córdoba, SpainServicio de Nefrología, Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Barcelona, SpainServicio de Nefrología, Hospital Virgen de la Candelaria, Santa Cruz de Tenerife, SpainServicio de Nefrología Pediátrica, Hospital La Fe, Valencia, SpainServicio de Nefrología, Hospital Universitario 12 de Octubre, Madrid, SpainEnfermedades Renales Hereditarias, Fundació Puigvert, Barcelona, SpainServicio de Nefrología, Complejo Hospitalario A Coruna, A Coruna, SpainDepartamento de Medicina Celular y Molecular, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain

a r t i c l e i n f o

rticle history:

eceived 9 April 2015

ccepted 3 July 2015

vailable online 3 December 2015

eywords:

typical haemolytic uraemic

yndrome

a b s t r a c t

Haemolytic uraemic syndrome (HUS) is a clinical entity defined as the triad of nonimmune

haemolytic anaemia, thrombocytopenia, and acute renal failure, in which the underlying

lesions are mediated by systemic thrombotic microangiopathy (TMA). Different causes can

induce the TMA process that characterises HUS. In this document we consider atypical

HUS (aHUS) a sub-type of HUS in which the TMA phenomena are the consequence of

the endotelial damage in the microvasculature of the kidneys and other organs due to a

disregulation of the activity of the complement system. In recent years, a variety of aHUs-

related mutations have been identified in genes of the complement system, which can

culizumab

omplement

hrombotic microangiopathy

explain approximately 60% of the aHUS cases, and a number of mutations and polymor-

phisms have been functionally characterised. These findings have stablished that aHUS is a

consequence of the insufficient regulation of the activation of the complement on cell sur-

faces, leading to endotelial damage mediated by C5 and the complement terminal pathway.

� Please cite this article as: Campistol JM, Arias M, Ariceta G, Blasco M, Espinosa L, Espinosa M, et al. Actualización en síndrome hemolíticorémico atípico: diagnóstico y tratamiento. Documento de consenso. Nefrologia. 2015;35:421–447.∗ Corresponding author.

E-mail address: JMCAMPIS@clinic.ub.es (J.M. Campistol).013-2514/© 2015 Sociedad Espanola de Nefrología. Published by Elsevier España, S.L.U. This is an open access article under the CCY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

422 n e f r o l o g i a. 2 0 1 5;3 5(5):421–447

Eculizumab is a monoclonal antibody that inhibits the activation of C5 and blocks the gen-

eration of the pro-inflammatory molecule C5a and the formation of the cell membrane

attack complex. In prospective studies in patients with aHUS, the use of Eculizumab has

shown a fast and sustained interruption of the TMA process and it has been associated

with significative long-term improvements in renal function, the interruption of plasma

therapy and important reductions in the need of dialysis. According to the existing literature

and the accumulated clinical experience, the Spanish aHUS Group published a consensus

document with recommendations for the treatment of aHUs (Nefrologia 2013;33[1]:27–45).

In the current online version of this document, we update the aetiological classifica-

tion of TMAs, the pathophysiology of aHUS, its differential diagnosis and its therapeutic

management.

© 2015 Sociedad Espanola de Nefrología. Published by Elsevier España, S.L.U. This is an

open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Actualización en síndrome hemolítico urémico atípico: diagnóstico ytratamiento. Documento de consenso

Palabras clave:

Síndrome hemolítico urémico

atípico

Eculizumab

Complemento

Microangiopatía trombótica

r e s u m e n

El síndrome hemolítico urémico (SHU) es una entidad clínica definida por la tríada ane-

mia hemolítica no inmune, trombocitopenia e insuficiencia renal aguda, en la que las

lesiones subyacentes están mediadas por un proceso de microangiopatía trombótica (MAT)

sistémico. Distintas causas pueden desencadenar el proceso de MAT que caracteriza el

SHU. En este documento consideramos SHU atípico (SHUa) como el subtipo de SHU en

el que los fenómenos de MAT son fundamentalmente consecuencia del dano producido

en el endotelio de la microvasculatura renal y de otros órganos por desregulación de la

actividad del sistema del complemento. En los últimos anos se han identificado diversas

mutaciones en genes del sistema del complemento asociados a SHUa, que explicarían

aproximadamente el 60% de los casos de SHUa, y se han caracterizado funcionalmente

numerosas mutaciones y polimorfismos asociados a SHUa que han permitido determinar

que la patología se produce como consecuencia de la deficiente regulación de la activación

del complemento sobre las superficies celulares y que lleva al dano endotelial mediado por

la activación del C5 y de la vía terminal del complemento. Eculizumab es un anticuerpo

monoclonal humanizado que inhibe la activación del C5, bloqueando la generación de la

molécula proinflamatoria C5a y la formación del complejo de ataque de membrana. En

estudios prospectivos en pacientes con SHUa su administración ha demostrado la inter-

rupción rápida y sostenida del proceso de MAT, con una mejora significativa de la función

renal a largo plazo y una reducción importante de la necesidad de diálisis y el cese de

la terapia plasmática. En función de las evidencias científicas publicadas y la experiencia

clínica acumulada, el Grupo Espanol de SHUa publicamos un documento de consenso con

recomendaciones para el tratamiento de la enfermedad (Nefrología 2013;33(1):27–45). En la

presente versión online del documento se actualizan los contenidos sobre la clasificación

etiológica de las MAT, la fisiopatología del SHUa, su diagnóstico diferencial y su manejo

terapéutico.

© 2015 Sociedad Espanola de Nefrología. Publicado por Elsevier España, S.L.U. Este es un

artículo Open Access bajo la licencia CC BY-NC-ND

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Haemolytic uraemic syndrome (HUS) is a clinical entity

consisting of the triad of nonimmune microangiopathichaemolytic anaemia, thrombocytopenia, and acute renalfailure.1 The histological lesions of HUS typically involve

systemic thrombotic microangiopathy (TMA), largely resultingin impaired intrarenal vessels. A greater number of HUS casesare caused by a Shiga toxin-producing (STEC) Escherichia colienteric infection or verotoxin-producing (VTEC) germs,

resulting in the so-called typical HUS or STEC (VTEC)-HUS.Genetic or acquired (autoantibodies) dysregulation of thealternative complement pathway leading to endothelial

1 5;3 5

d1c(

iaaipopeamap

cieoept

and Metalloproteinase with ThromboSpondin type 1 motif, mem-ber 13 (ADAMTS13), a plasma enzyme responsible for splitting

FMc

n e f r o l o g i a. 2 0

amage and systemic TMA phenomena occur in nearly0% of HUS reports.2 This kind of HUS related to theomplement dysregulation is known as atypical HUSaHUS).

In 2011, Eculizumab (Soliris®; Alexion Pharmaceut-cals, Connecticut, USA) was approved by the Americannd European regulatory agencies for the treatment ofHUS.3 Eculizumab is a humanised monoclonal antibodynhibiting C5 activation and blocking the production of theroinflammatory C5a anaphylatoxin, as well as the formationf the membrane-attack complex, leading to cell lysis.4 Inrospective studies conducted in aHUS patients, Eculizumabffectively halted the TMA process and its effects, and wasssociated with the rapid, significant, and long-term improve-ent of haematological and renal function abnormalities,5

nd with improved systemic involvement and high bloodressure.

In 2012, the Spanish Group for aHUS gathered to develop aonsensus document including recommendations for treat-ng the disease.6 The group has been meeting every yearver since then in order to update both the understandingf the various aspects of interest related to the dis-

ase (including the aetiological classification of TMAs, theathophysiology of aHUS, and companion diagnostics) andreatment recommendations based on already published

ig. 1 – Renal histopathological lesions from haemolytic uraemicesangiolysis (C) Thrombi in the glomerular capillaries (arrow).

ourtesy of Dr. R. Ortega (Histopathology department of the Hosp

(5):421–447 423

scientific evidence and clinical experience. The contents orig-inally published in Nephrology 2013;33(1):27–45 have beenupdated in the current online version of the consensusdocument.

Aetiological classification of thromboticmicroangiopathies

The term TMA describes a histological lesion of the arte-rioles and capillaries resulting in thickened and swollenvessel walls, detachment of endothelial cells, widening ofthe subendothelial space caused by the build-up of proteinsand cell lysis material, and the presence of platelet thrombiobstructing vascular lumen (Fig. 1).1 Two clinical entities withdifferent aetiology and pathophysiology are characterised byprimary TMA lesions: thrombotic thrombocytopenic purpura(TTP) and HUS.

Intravascular thrombosis in TTP results from a severe defi-ciency in the metalloprotease activity of the A Desintegrin

the ultra-large multimers of the Von Willebrand factor.7 Thisdeficiency may be genetic or acquired via IgG circulating

syndrome. (A) Ischaemic and retracted glomeruli. (B)(D) Artery occluded by platelet thrombi. Photographsital Universitario Reina Sofía, Córdoba).

0 1 5

incidence of aHUS in the US has been estimated to be of36

424 n e f r o l o g i a. 2

antibodies blocking ADAMTS13 (particularly in patientsreceiving platelet antiaggregants).8

Ninety percent of HUS cases are caused by a STEC entericinfection resulting from contaminated food (typical HUS orSTEC [VTEC]-HUS).2 The Shiga toxin causes a direct injuryon the vascular endothelium, triggering a number of celland vascular events which ultimately lead to TMA.2 Clinicalpresentation usually involves abdominal pain and diarrhoea,together with acute renal failure within 4–10 days. Prognosisis typically favourable, with a mortality rate below 5% and 80%of patients achieving complete clinical recovery, although pro-gression to severe chronic renal failure is observed over timein up to 20–30% of patients.9,10

aHUS is essentially diagnosed by exclusion onceADAMTS13 (TTP) deficiency or STEC infection (STEC-HUS)are ruled out. In patients with aHUS, TMA phenomenaare a consequence of the dysregulation of the alternativecomplement pathway on the cell surface. This abnormal-ity results in uncontrolled activity on own cells followingcomplement activation (by several triggering factors), lead-ing to endothelial damage, inflammation, and secondarythrombosis, with an increasing number of cases involvinggenetic or acquired factors. Mutations have been describedin one or more complement proteins in nearly 60% of over1000 patients with reports of aHUS in the literature,11–18

although a genetic component (involving complement genesor other, including coagulation genes) and/or unspecifiedautoimmunity may also be present in the remaining patients.Of note, anti-Factor H (FH) autoantibodies have been foundin 5–10% of aHUS patients.19 Unlike STEC-HUS, which isusually an isolated event, aHUS is a chronic and relaps-ing entity triggered by the uncontrolled activation of thecomplement system. Before Eculizumab became available,aHUS had been mostly associated with a poor prognosis: themortality rate following a first episode of aHUS was 10–15%,and renal function remained unrecovered in up to 50% ofpatients.11,12,20

A type of aHUS resulting from recessive mutations in theDGKE gene coding for the DGK-� (diacylglycerol kinase-�) pro-tein has recently been described.21 The loss of this enzymeactivity in endothelial cells, platelets, and podocytes leads toendothelial cells apoptosis and impaired angiogenic response,thereby resulting in a prothrombotic and inflammatorystate.22 Patients with DGKE mutations exhibit various pheno-types ranging from aHUS to membranoproliferative glomeru-lonephritis with high proteinuria and nephrotic syndrome.23

aHUS patients develop persistent high blood pressure andhaematuria-proteinuria (including in the nephrotic range) intheir first year of life. Unlike paediatric aHUS associated withcomplement genetic alterations, progression to chronic renaldisease among these patients is not sudden, but develops overyears.21

In addition to STEC enteric infection (typical HUS),abnormalities in the regulation of complement activation,mutations in DGKE or coagulation genes (aHUS), or (geneticor autoimmune) deficiency of ADAMTS13 in TTP, there aremany other factors and clinical entities that may be asso-

ciated with the development of TMA. This kind of TMAis included under the term secondary TMA. Some casesreported in children are associated with methylmalonic

;3 5(5):421–447

aciduria24 or more commonly (5% of HUS reports in chil-dren) with neuraminidase-producing invasive Streptococcuspneumoniae infections (resulting in the exposure of the crypto-antigen T in the cell surface and unleashing the TMAphenomenon),25 or H1N1 infection.26 TMA has been gener-ally associated with viral infections (CMV, HIV, parvovirus),neoplastic processes, drugs (antitumor agents, including thevascular endothelial growth factor inhibitors, immunosup-pressants such as calcineurin inhibitors [cyclosporine andtacrolimus] or the mammalian target of rapamycin inhibitors[mTOR; sirolimus, everolimus], platelet antiaggregants, antivi-rals, or oral contraceptive drugs), malignant high bloodpressure, bone marrow or solid organ transplantation, preg-nancy and postpartum, autoimmune systemic diseases, orglomerulonephritis.27

Importantly, the foregoing causes of TMA may not alwaysbe identified in all patients, whereas some may presentmore than one aetiology, resulting in a heterogeneous pre-sentation and a challenging diagnosis. In fact, overlappingentities are common and up to 25% of patients with STEC-HUS and 86% of patients with pregnancy-associated HUSdevelop complement system mutations, where aHUS is actu-ally the underlying disease.28,29 Mutations in the complementsystem have also been reported in post-transplant HUS asso-ciated with the use of calcineurin inhibitors and in HUSrelated to autoimmune diseases in 27% and 33% of patients,respectively.12 Furthermore, several cases of secondary TMAhave been reported to date with successful treatment out-comes with Eculizumab (TMA associated with drugs,30 solidorgans31 or bone marrow32 transplantation, pregnancy33 andsystemic erythematous lupus34). The fact that complementblockade (by Eculizumab) is associated with a favourableclinical response and the reversibility of TMA suggests thepotential and important role of non-genetic complement dys-regulation in many cases of secondary TMA, predisposingpatients to its development. On this basis, Fig. 2 sum-marises the proposed aetiological classification of TMAs andillustrates the potential overlapping between these clinicalentities. The classification of TMAs should be understoodas a current topic of interest and major debate is tak-ing place among the medical community as a result ofthe continuous progress made in the understanding of thepathophysiology of these entities.35 Given that the aHUSmediated by complement dysregulation is the main reasonfor debate, only this entity will be discussed in the followingsections.

Atypical haemolytic urinary syndrome:a clinical entity

Epidemiology

aHUS is considered an ultra-rare disease. Data available on itsincidence and prevalence are limited, as well as the knowl-edge of the actual epidemiology of the disease. The annual

∼1–2 cases/million inhabitants. A recent multicentre studyin Europe reported an incidence of 0.11 cases/million inhabi-tants. According to the European Medicines Agency (EMA), the

n e f r o l o g i a. 2 0 1 5;3 5(5):421–447 425

TTP (ADAMTS13 activity <5-10%- Genetic cause- Antibodies

TTP

TMAsecondaries

aHUS(associated with

complement)

Shiga toxin-producing Escherichia coli- Strain 0157:H7 and other strains- Shigella disenteriae type I- Streptococcus pneumoniae (neuraminidase)

Complement abnormalities- Mutations in FH, MCP, FITHBD, FB and C3- Polymorphism risk in FH and MCP- Anti-FH antibodies

STEC- HUS and STEC-like

Pregnancy - Pre-eclampsia and eclampsia - Anti-tumour- HELLP syndrome - Mitomycin C.- Postpartum TMA Gemcitabine, CisplatinSystemic diseases - VEGF and tyrosine kinase - SLE inhibitors (sunitinib,- Vasculitis imatinib and dasatinib)- Scleroderma - Everolimus- Anti-phospholipid syndrome - RadiationGlomerulonephritis - Others - C3 Glomerulopathies - Quinine - Glomerulopathies associated with - Interferonmonoclonal gammapathies - Ticlopidine and clopidogrelof uncertain significance - Valaciclovir- IgA nephropathy - Oral contraceptivesMalignant arterial hypertension Bone marrow transplant Infections Solid organ transplant- HIV - Drugs- HCV Immunosuppressives- H1N1 (influenza A) - Humoral rejection - OthersNeoplasms Drugs/treatments - Immunosuppressives - Calcineurin inhibitors (cyclosporine, tacrolimus) - mTOR inhibitors (sirolimus, everolimus)

- Viral infections (CMV and BK virus) Other causes of TMA - Methylmalonic aciduria - Intestinal lymphangiectasis

Fig. 2 – Classification of the aetiologies of thrombotic microangiopathies. ADAMTS13: A Disintegrin And Metalloproteinasewith a ThromboSpondin type 1 motif, member 13; aHUS: atypical haemolytic uraemic syndrome; CMV: cytomegalovirus; FB:complement factor B; FH: complement factor H; FI: complement factor I; HCV: hepatitis C virus; HELLP: Hemolysis, ElevatedLiver enzymes, Low Platelet count; HIV: human immunodeficiency virus; HUS: haemolytic uraemic syndrome; MCP:membrane cofactor protein; mTOR: mammalian target of Rapamycin; SEL: systemic erythematous lupus; STEC: Shigatoxin-producing Escherichia coli; THBD: thrombomodulin; TMA: thrombotic microangiopathy; TTP: thrombocytopenicthrombotic purpura; VEGF: vascular endothelial growth factor.

paa

adsa

C

Cdssttoah

revalence may be of ∼3.3 patients/million inhabitants/yearmong patients below the age of 18, with lower rates amongdults.

Children and adults are predominantly affected by aHUS,lthough it may develop at any time in life.11,12 The onset of theisease usually occurs before the age of 18 (60% vs. 40%) andex characteristics are well-balanced (women are primarilyffected when the disease is developed in adulthood).11,13

linical presentation

linical onset is often abrupt, although 20% of patients mayevelop it progressively (in weeks or months), accompanied byubclinical anaemia, fluctuating thrombocytopenia, and pre-erved renal function.11 The clinical picture comprises theriad of nonimmune microangiopathic haemolytic anaemia,

1

hrombocytopenia, and acute renal failure. High levelsf lactate dehydrogenase (LDH), undetectable haptoglobinnd schistocytes confirm the presence of intravascularemolysis20 associated with haematuria, proteinuria, and/or

acute renal failure (with or without oligoanuria). High bloodpressure resulting from volume overload or vascular lesion iscommon.1 In some patients, the single manifestation of TMAmay be proteinuria with high blood pressure and progressiverenal failure without haematological abnormalities.

Even though aHUS lesions are predominantly observedin renal vessels, the diffuse and systemic nature of theTMA phenomenon leads to the involvement of the microvas-culature of other organs (including, but not limited tothe brain, heart, intestine, pancreas, and lungs),1 thereforeaccounting for common extrarenal symptoms.11,12 Neuro-logical symptoms are the most common (48%),37 includingirritability, somnolence, confusion, convulsions, encephalo-pathy, stroke, hemiparesis, visual abnormalities, hemiplegia,and coma.1,12,37,38 Myocardial infarction has been describedin up to 3% of aHUS patients in relation to sudden death.12,39

Myocardiopathy, heart failure, and peripheral ischaemicvasculopathy have also been reported,19,37,40,41 as well asdiarrhoea (30%) and other digestive symptoms (including,but not limited to colitis, nausea, vomit, abdominal pain,

0 1 5;3 5(5):421–447

Table 1 – Risk factors in atypical uraemic haemolyticsyndromea

MutationsLoss of function

CFH (∼13%)MCP (∼11%)CFI (∼10%)THBD (∼4%)

Gain of functionC3 (∼4%)CFB (∼3%)

PolymorphismsIncreasing risk

CFH: c.-332C>T; c.2016A>G (p.Gln672Gln); c.2808G>T(p.Glu936Asp)

MCP: c.-652A>G; c.-366A>G; c.989-78G>A; *897T>CProviding protection

CFH: c.184G>A (p.Val62Ile)Autoantibodies

Anti-FH (∼5%)Environmental factors

InfectionsImmunosuppressantsOral contraceptivesAnti-cancer drugs

Anti-FH: anti-complement factor H antibody; CFB: complement fac-tor B gene; CFH: complement factor H gene; CFI: complement factorI gene; MCP: membrane cofactor protein. Gene; THBD: thrombomo-dulin gene.a “Multiple hits” theory. aHUS is a complex disease normally involv-

ing various risk, genetic, and environmental factors. Patients arecommonly carriers of more than one mutation in complementgenes or combined mutations with risk polymorphisms. Envi-ronmental factors are also necessary to help reveal the geneticdisposition from mutations or polymorphisms. Concomitantmutations with risk polymorphisms, autoantibodies, or trigger-ing environmental factors account for the incomplete penetranceof aHUS, as well as for the differences in its presentation andprogression among carriers of complement gene mutations.

426 n e f r o l o g i a. 2

hepatitis, cholestasis, and pancreatitis).12,19,38,42 Skin involve-ment including ulcer lesions in lower limbs has recently beenreported in aHUS patients.43 Heterogeneity of symptoms hasposed a challenge for companion diagnostics of other causesof TMA.

Pathophysiology

The complement system, consisting of several circulatingplasma and membrane-associated proteins, is part of innateimmunity and is vital for fighting infections, processingimmune complexes, antibody response, and the elimina-tion of apoptotic residues. Activation by any of the existingpathways (classical, lentin, and alternative) leads to the for-mation of multiprotein complexes with C3-convertase activitysplitting the C3 protein and resulting in C3b (Fig. 3). Thecovalent binding of this molecule to the surfaces activatingthe complement favours phagocytosis by polymorphonuclearleukocytes and macrophages, therefore resulting in activatedC5, directing the attack complex to the membrane and caus-ing cell lysis. In addition, the resulting C3b leads to a rapidlyenhanced complement activation by promoting the formationof further C3-convertases, since it is one of the compo-nents of C3-convertase of the alternative pathway.44 In orderto avoid total uptake by complement activation, as well asdamage to self tissues (C3b binds indiscriminately to bothpathogens and self cells), a number of process-regulatingproteins, such as FH, the membrane cofactor protein (MCP),and complement factor I (FI) dissociate C3-convertases andresult in C3b degradation. C3b levels, therefore, remain lowunder normal conditions and they build up following com-plement activation only in the structures related to thisactivity.

Several studies have shown that around 60% of aHUSpatients are carriers of mutations in complement-regulatinggenes (CFH, MCP, CFI, thrombomodulin [THBD], or in the com-ponents of C3-convertase, factor B [FB], and C3).45–54 All thesemutations cause the dysregulation of the alternative pathway(Table 1). FH acts in plasma by controlling complementhomeostasis and in cell surfaces by preventing damage to selfcomponents. Mutations in the C-terminal region of FH arecharacteristic of aHUS. Because the altered FH region medi-ates complement activation in cell surfaces, cell protectionagainst accidental damage resulting from complement acti-vation is decreased by these mutations, with no involvementof complement regulation in plasma.55 The functional assayof aHUS-associated mutations found in other complementgenes, including MCP, CFI, CFB or C3, has also confirmed thatall of them result in a defective protection of cell surfacesand this loss of complement regulation may be due to thedecreased activity of regulating proteins or to the abnormallyhigh activity of C3-convertases. Thus, while the regulatoryactivity of these proteins is impaired by mutations in FH, MCP,and FI, mutations in FB or C3 result in further activation of theC3-convertase.

Around 5–10% of aHUS patients develop anti-FH antibodiestargeted to the C-terminal region, with similar effects to thoseobserved in FH mutations.56,57 Their role in the pathogenesisof aHUS has not been fully established, but seems to be

associated with disease onset or recurrence. Given that anti-body titres may decrease over time, they should be screenedearly in the course of aHUS. Anti-FH antibodies are associatedwith complement factor H-related protein 1 deficiency (FHR1)in patients with aHUS.58

Penetrance of aHUS in carriers of mutations in some genesis around 50%, with only a few carriers from families withidentified mutations commonly developing aHUS and show-ing a variable clinical presentation. Clinical heterogeneity,which results from the existence of additional (genetic andenvironmental) risk factors mediating the development andthe outcome of the disease, is largely observed among unre-lated carriers of this mutation. Screening for complementmutations in aHUS patients and conducting case-control stud-ies based on genetic polymorphisms in candidate genes orgenetic markers in the human genome has allowed for theidentification of some variants (polymorphisms) in CFH andMCP genes modulating the penetrance and severity of the dis-

ease (Table 1).49,59,60

Haplotypes CFH-H3 and MCPggaac are the most rele-vant polymorphisms associated with the risk of aHUS. Both

n e f r o l o g i a. 2 0 1 5;3 5(5):421–447 427

Classic andlectins pathway

C3-convertase (C4b2a)

C3-convertase(C3bBb)

Alternativepathways

aHUS mutations

Cellmembrane

Eculizumab

C3

C3b

(formation)

(activation)(C5-convertase)

MACC5-9

FHMCP

FI

C3b

FBC3

C5

Lytic pathway

Fig. 3 – Complement dysregulation in atypical haemolytic uraemic syndrome. Complement activation by any of the 3pathways (detection of foreign antigens, alternative pathway; of antibodies, classical; or mannan polysaccharides, lectin)leads to the build-up of large quantities of C3b on the activator cell membrane, causing opsonisation and C5 activation(terminal or lytic pathway), resulting in the formation of the membrane attack complex and cell lysis. Complementactivation results in inflammation and leucocyte recruitment. The key process in complement activation is C3b formation,which depends on unstable enzymatic complexes – C3-convertases – catalysing the rupture of C3 to create C3b. In turn, C3bhas the ability to form further C3-convertase of the alternative pathway (C3bBb), thus enhancing the initial activation. Themediation of C3B production is two-fold: dissociation of C3-convertases and proteolytic inactivation of C3b and C4b. Severalregulatory proteins in plasma and the cell membrane carry out this regulatory activities, including, factor H, MCP and factorI, which play an essential role in the dissociation of C3-convertase of the alternative pathway (C3bBb) and the proteolyticdegradation of C3b. Mutations in these proteins found in patients with aHUS interfere with this regulatory activity of thealternative pathway activation. Some patients with aHUS are carriers of mutations in proteins C3 and factor B organisingC3-convertase. These mutations are particular, as they increase the activity of mutated proteins (gain-of-functionmutations), resulting in increased complement activation and exceeding the capacity of regulatory proteins.

htaswAopcthitp

toocs

aplotypes include single-nucleotide polymorphisms (SNP) inhe CFH and MCP gene-promoter region, downregulating FHnd MCP. The presence of both polymorphisms in homozygo-is may provide a rationale for aHUS disposition in patientsith no mutations in any of the genes associated with aHUS.

recent collaborative study by the European Working Partyn Complement Genetics in Renal Diseases including 795atients with aHUS has shown that 3% of these patients werearriers of combined mutations in more than one gene. Addi-ionally, this large study has proved that concomitant riskaplotypes CFH-H3 and MCPggaac also lead to significantly

ncreased disease penetrance in carriers of combined muta-ions, stressing the idea that genotyping of these risk polymor-hisms helps predict the risk of aHUS in mutation carriers.61

Along with the previous genetic alterations, a number ofriggering environmental factors are also implicated in thenset of aHUS. The above mutations are predisposing factors

f the disease, preventing adequate complement regulation inell surfaces when the system becomes activated in microves-els. aHUS is triggered by infectious events in 50–80% of

patients,11,12,40 particularly those involving the upper respi-ratory tract (influenza H1N1 virus). Diarrhoea caused by gas-troenteritis may precede aHUS in up to 30% of cases (includingdiarrhoea by STEC11,12,19).12 Pregnancy, particularly duringpost-partum, is a common predisposing factor of aHUS amongwomen,12,29 together with the use of oral anovulatory agents.

Mutations in the gene coding for thrombomodulin (THBD),an anticoagulant protein acting as thrombin cofactor and alsoregulating the FI-mediated C3b inactivation, have been associ-ated with aHUS.62 Based on complement dysregulation typicalof patients with aHUS, the functional analysis of THBD muta-tions associated with aHUS has shown that thrombomodulinmutations impair the complement regulatory activity.62 Nev-ertheless, the impairment of the anticoagulation activity bythrombodulin mutations associated with aHUS and the rel-evance of these abnormalities in aHUS remains unknown.In this regard, a recent study conducted in 36 patients with

aHUS has assessed the presence of mutations in the genesof the complement system and coagulation through mas-sive DNA sequencing, detecting mutations in genes from both

428 n e f r o l o g i a. 2 0 1 5;3 5(5):421–447

Table 2 – Clinical outcome of patients with atypical haemolytic uraemic syndrome based on complement abnormalities(prior to Eculizumab).

Gene Risk of death or ESRF inthe first episode or

within the next year

Risk of relapse Risk of death or ESRF at3–5 years

Risk of relapse followingrenal transplant

CFH 50–70% 50% 75% 75–90%CFI 50% 10–30% 50–60% 45–80%MCP 0–6% 70–90% 6–38%a <20%C3 60% 50% 75% 40–70%CFB 50% 3/3 without ESRF 75% 100%THBD 50% 30% 54%a 1 patientAnti-FH 30–40% 40–60% 35–60%a Higher with increased

antibody titres

Anti-FH: anti-complement factor H antibodies; CFB: complement factor B gene; CFH: complement factor H gene; CFI: complement factor I gene;ESRF: end-stage renal failure; MCP: membrane cofactor protein gene; THBD: thrombomodulin gene.a Data on ESRF.

Adapted from Loirat and Fremeaux-Bacchi.1

systems.63 The gene in the coagulation system with the largestnumber of mutations was plasminogen (PLG), a cymogenewhich plays an important role in fibrinolysis following con-version to plasmin. Even though these data suggest that coag-ulation genes may add to aHUS disposition (particularly PLG),further studies are required to confirm these observations.

The search of new genes associated with aHUS has alsobeen addressed by Lemaire et al.21 through exome sequenc-ing. The authors have identified homozygotic mutations in theDGKE gene coding for the DGK-� protein in 13 patients withaHUS from 9 families. These patients had a very early onsetof aHUS, generally within their first year of life, followed bymultiple recurrences and common progression to end-stagerenal failure in the second decade of life.21 Deficiency of DGK-� in endothelial cells has been recently shown to induce theexpression of ICAM-1 and tissue factor by means of increasedp36-MAPK-mediated signalling, leading to apoptosis, alteringthe angiogenic response, and determining a proinflammatoryand prothrombotic phenotype. Yet, the absence of DGK-� isnot detrimental to complement activation in cell surfaces.21,22

The absence of DGK-� in podocytes and endothelial cellsmay probably impair the diaphragm of glomerular filtration,which would account for massive proteinuria and the sus-ceptibility to glomerular conditions among these patients,21,23

although the reason why these patients tend to develop sev-eral glomerular conditions remains uncertain. Finally, despitethe role of the complement in the development of renal dis-ease among carriers of DGKE, mutations had been initiallyruled out,21 patients with DGKE mutations additionally asso-ciated with other genes previously related to aHUS, includingTHBD and C3,64 have been recently identified, thus suggestingthat complement dysregulation may play a role in the modu-lation of disease onset and outcome at least in some carriersof DGKE mutations.

Prognosis

The availability of Eculizumab has significantly revolutionisedthe prognosis of patients living with aHUS, a very severe

disease in most cases in spite of intensive treatment withplasma therapy (PT; Table 2). Following a first episode of aHUS,overall mortality was higher than 10% and more than halfof the patients required dialysis and/or developed a morepermanent renal damage in the next 12 months.11,12,20 Theclinical outcome changes relatedly depending on the patient’smutation. In this respect, outcome seemed to be particularlypoor in patients with FH and C3 mutations, with mortalityand end-stage chronic renal failure (ESCRF) rates over 50%within one year from the first episode of aHUS. Furthermore,half of these patients relapsed. Mutations in FI, FB, and THBDwere also associated with high rates of mortality and ESCRFat one year (50%), with relapses occurring in nearly one in3 patients overcoming the first episode of aHUS. On theother side, less than 10% of patients with MCP mutationsdied or progressed to ESCRF, although the risk of relapseamong these patients was higher and up to 90% of themdeveloped new episodes of aHUS. Between 50 and 75% ofpatients with mutations in FH, CI, C3, FB or THBD died ordeveloped ESCRF within 3–5 years from the first episode ofaHUS.1

Recurrence of atypical haemolytic uraemic syndromefollowing renal transplantation

The outcome of renal transplantation (RT) among patientswith ESCRF due to aHUS has been historically limited by theincreased percentage of recurrences of post-transplant dis-ease (∼50%; graft loss rate: 80–90%65,66), although it changessignificantly based on the type of alteration. In a series of 57patients with aHUS receiving RT, 5-year recurrence-free graftsurvival was significantly lower in patients with mutationsin the genes coding for complement proteins comparedto patients in whom only polymorphisms but no geneticabnormalities were found.67 Yet, it should be stressed that

the risk of recurrence of aHUS following RT in patients withno genetic abnormalities is also deemed high. 68 Mutationsin FH are associated with a higher risk of recurrence orgraft loss following RT (75–90%; specifically those related to

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Table 3 – Diagnostic tests and procedures recommended for patients with thrombotic microangiopathy.

General diagnostic tests• Complete medical records, including drugs, data from systemic diseases, personal and family history• Complete physical examination, including a fundoscopic exam• General routine blood and urine tests• Haptoglobin levels• Serum complement levels• Peripheral blood smear• Serology for systemic diseases (ANA, anti-ADN, ANCA, antic-Scl-70, anticentromere)• Anti-cardiolipin antibodies and lupus anticoagulant• Serology for HIV, HCV, HBV, CMV and H1N1• Complete clotting test, with fibrinogen, fibrinogen degradation products and dimer D• Investigations for typical HUS-causing bacterial infections and Shiga toxin test (if clinically suspected)

Specific diagnostic tests

• STEC infection • Faecal sample in case of diarrhoea or rectal culture: STEC cultures (MacConkey for E. coli O157:H7); PCR for Stx genes O157:H7 and otherserotypes, and other virus characteristics; ELISA and/or Vero cell tissue culture assay for Stx serum: anti-LPS antibodies for prevalent serotypes

• Pneumococcal infection • Bacterial culture (generally) of sterile body fluids; DAT (Coombs test), viral test (respiratory), chest x-ray (pleural effusion as a characteristic inmost cases), cytochemistry, and CSF culture in cases to pneumococcal meningitis

• Altered regulation of the complement • C3, C4 (plasma/serum), AH50• FH, FI, FB (plasma/serum)• Anti-FH autoantibodies• Expression of superficial MCP in leukocytes (poly- or mononuclear leukocytes using a FACS test)• Mutation analysis in FH, FI, MCP, C3, FB ± THBD

• ADAMTS13 deficiency (acquired or hereditary) • Plasma activity of ADAMTS13 or dose (ELISA) ± inhibitor• Cobalamin metabolism: methylmalonic aciduria • Amino acid chromatography in plasma/urine (hyperhomocysteinemia, hypomethioninemia; homocystinuria); organic acid chromatography in

urine (methylmalonic aciduria)• Mutation analysis for the gene MMACHC

ADAMTS13: A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13; ANA: antinuclear antibody; ANCA: Autoantibodies to neutrophil cytoplasmic antigens; CMV:cytomegalovirus; CSF: cerebrospinal fluid; DAT: direct antiglobulin test; DNA: deoxyribonucleic acid; ELISA: enzyme-linked immunoabsorption assay; FACS: fluorescence activated cell sorting; FB:complement factor B; FH: complement factor H; FI: complement factor I; HIV: human immunodeficiency virus; HUS: haemolytic uraemic syndrome; MCP: membrane cofactor protein; STEC: Shigatoxin-producing Escherichia coli; THBD: thrombomodulin; VHB: hepatitis B virus; VHC: hepatitis C virus.Specific diagnostic tests: adapted from Loirat et al.2

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Table 4 – Differential diagnosis between disseminatedintravascular coagulation and thromboticmicroangiopathy.

DIC TMA

Platelet count ↓ ↓Fibrinogen ↓ NormalFibrinogen degradation products ↑ NormalDimer D ↑ NormalAntithrombin ↓ NormalSchistocytes Present PresentHaptoblogin Normal ↓Coagulation times Long NormalBlood pressure ↓ ↑

DIC: disseminated intravascular coagulation; TMA: thromboticmicroangiopathy.

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abnormalities in terminal 3′ and gene conversion betweenCFH and CFHR1, resulting in the hybrid gene CFH/CFHR1 [bothabnormalities impair the functionality of the C-terminaldomain of FH]), posing a high risk with mutations in C3and FI as well (40–80%; Table 2).12,42,48,65,67,69–71 To date, veryfew transplants have been performed in patients with FBmutations, though recurrence of aHUS and graft loss werereported in all cases.49,72 In general, plasma factors of thecomplement involved in aHUS are synthesised in the liver,and so patients with mutations in the complement genescoding for these factors remain prone to aHUS following RT,as dysfunctional factors are still being produced. MCP is atransmembrane protein that is highly expressed in the kidneyand, as a result, this defect can be corrected by RT by deliver-ing unchanged MCP into the graft. Over 80% of patients withMCP mutations develop no recurrence of aHUS following RT,with a long-term survival rate comparable to that of patientsreceiving transplants for other reasons.40,65,66 The risk of post-transplant recurrence in patients with THBD62 mutations oranti-FH antibodies is not well-established, although it seemsto be related to high and persistent titres of antibodies in thelatter.19,73

Diagnosis of atypical haemolytic uraemicsyndrome

In light of the rapid evolution and the severity of TMA, adifferential diagnosis should be immediately establishedfrom the syndrome perspective, allowing for supportive mea-sures to be taken within 24–48 h from patient’s admission.Considerations for an aetiological diagnosis of TMA will sub-sequently be made. Table 3 summarises the major proceduresand diagnostic tests recommended for the diagnosis of TMA,including specific tests for companion diagnostics of thevarious aetiologies of TMA.

In patients with TMA, tests results include thrombo-cytopenia (platelet count <150,000/mm3 or decrease >25%from baseline)20 and microangiopathic haemolytic anaemia(haemoglobin <10 mg/dl with a negative direct Coombs test[though some patients with pneumococcal or H1N1-relatedHUS may show positive direct Coombs test],25 elevated LDH,decreased haptoglobin, reticulocytosis, and schistocytes).20,62

In a retrospective series of 50 patients with histological TMA,44% had a normal platelet count.74 Consequently, diagno-sis of TMA should be considered in patients with renalfailure and elevated LDH, but without thrombocytopenia.For schistocytes, even though they can be found in mostpatients with renal disease, preeclampsia or mechanic valves,TMA can be diagnosed with a schistocyte count >1% pro-vided that there are no other known causes.75 In contrast,the absence of schistocytes does not rule out a diagnosisof TMA.

High levels of serum creatinine, low glomerular filtration(GF) or the presence of proteinuria and/or haematuria11,20,69

are indicative of renal failure. A renal biopsy may be required

for adult patients following acute renal failure to determinethe aetiology, rule out other processes, and assess progno-sis, although the indication and time for biopsies must beexamined individually in patients with suspected TMA due

to the risk of bleeding. In this sense, diagnostic renal biop-sies are not recommended in patients conclusively diagnosedwith aHUS (positive family history, recurrence, etc.). Amongpaediatric patients, the diagnosis is essentially made on thebasis of clinical presentation, though renal biopsy may occa-sionally be required (especially in cases of secondary TMA orRT). Patients with clinical suspicion of TMA should alwaysbe examined by a nephrologist in light of the urgent treat-ment strategy required to ensure minimum irreversible renaldamage.

Disseminated intravascular coagulation (DIC) is a syn-drome that may be associated with several major laboratoryand clinical findings related to TMA. DIC is characterisedby a systemic activation of coagulation, secondary to sev-eral clinical conditions (sepsis, trauma or certain tumours),leading to thrombosis and bleeding, commonly involvingrenal function.76 Key test criteria based on coagulation testsfor differential diagnosis of DIC and TMA are listed inTable 4.

A complete and detailed clinical history should be madefor TMA patients, including personal and family history, trigg-ering factors (drugs, infections), and a thorough physicalexamination. As opposed to previous considerations madeseveral years ago, signs and symptoms of the different typesof TMA are currently thought to be nonspecific and avoid thecompanion diagnostics between these two entities.1 The dif-ferentiation between HUS and TTP was classically based onclinical criteria, with HUS and TTP being diagnosed whenrenal involvement and neurological involvement were pre-dominant, respectively. However, 50% of patients with TTPdevelop renal failure and 50% of patients with aHUS developneurological abnormalities.37,77

Clinical features do not allow for a differentiation betweenSTEC-HUS and aHUS as well, given that up to 30% of aHUScases are developed following gastroenteritis12 or patientsdevelop diarrhoea42 (a typical symptom of STEC-HUS). Onthe other hand, platelet count and the severity of renalinvolvement can actually guide companion diagnostics. Over-

all, TTP presents with severe thrombocytopenia (<20,000/mm3

in 73% of patients with acquired TTP)78 and moderaterenal involvement, whereas aHUS usually presents with

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Thrombocytopenia<150.000 or>25% decrease ±

Microangiopathic haemolysisElevated LDH

Haptoglobin decreaseSchistocytes

Haemoglobin decrease*

Plus more than one of the following:

Evaluate ADAMTS13 and Shiga toxin /STEC** test

TTP

<5-10% ADAMTS13 activity >5-10% ADAMTS13 activity Shiga toxin /STEC positive

aHUS TMA secondaries STEC-HUS***

Neurological symptomsConfusion Seizures

Renal involvementElevated creatinine

Decrease in estimated glomerular filtrate Urinary changes

Gastrointestinal involvementDiarrhoea

Nausea/vomitingAbdominal painGastroenteritis

Fig. 4 – Algorithm for the differential diagnosis of primary thrombotic microangiopathy. ADAMTS13: A Disintegrin AndMetalloproteinase with a ThromboSpondin type 1 motif, member 13; aHUS: atypical haemolytic uraemic syndrome; HUS:haemolytic uraemic syndrome; LDH: lactate dehydrogenase; STEC: Shiga toxin producing Escherichia coli; TTP: thromboticthrombocytopenic purpura. * Negative direct Coombs test. ** The Shiga toxin test/STEC is indicated when the patient has ahistory of digestive involvement or gastrointestinal symptoms. *** STEC infection can rarely trigger the underlying diseaseactivity in some patients with aHUS.

mrmeSbcAtsrc

Tu

Tgm

oderate thrombocytopenia (50–100,000/mm3) and severeenal involvement. This rule can be deemed a guide, but deter-

ination of ADAMTS13 activity and the Shiga toxin test aressential for an accurate differential diagnosis between TTP,TEC-HUS, and aHUS (Fig. 4). The diagnosis of STEC-HUS cane confirmed by the presence of the Shiga toxin or a positiveulture of STEC in patients with TMA,28 while the activity ofDAMTS13 in plasma should be <5–10% in order to confirm

he diagnosis of TTP.79,80 The diagnosis of the remaining caseshould be directed towards aHUS,79 and so additional tests areequired to rule out secondary TMAs. Test samples should beollected prior to PT.

reatment options for atypical haemolyticraemic syndrome

reatment for aHUS should involve two different strate-ies: on one side, supportive treatment measures aimed atanaging the consequences of aHUS (acute renal failure,

high blood pressure, anaemia, thrombocytopenia, etc.), and atargeted therapy to halt and revert TMA. Specific options forthe management of aHUS will be reviewed in this section.

Plasma therapy

PT can be delivered as plasma infusion (PI) and plasmaexchange (PE). In PI, patients are given virus-inactivated,non-native fresh frozen plasma (FFP), adding functional com-plement regulators.81 In PE, a patient’s plasma is replacedwith FFP, which not only results in the administration ofhigh doses of complement-regulating proteins, but also inthe elimination of dysfunctional endogenous soluble comple-ment inhibitors, minimising the risk of volume overload. Inaddition, anti-FH antibodies are also cleared in PE, togetherwith potential inflammatory/thrombogenic factors involved

in endothelial damage and platelet hyperaggregation. Thetreatment of choice recommended for episodes of aHUS tra-ditionally consisted of early and intensive PE at high volumesand of variable frequency based on disease activity. PIs are

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Table 5 – Prognosis of patients with atypical haemolyticuraemic syndrome treated with plasma infusion orplasma exchange.

Remission Death orend-stage renal

failure

CFH 63% (complete: 5%;partial: 58%)

37%

CFI 25% (complete:12,5%; partial: 12,5%)

75%

C3 57% (complete: 43%;partial: 14%)

43%

THBD 88% (complete: 62%;partial: 25%)

13%

Anti-FH antibodies 75% (complete: 25%;partial: 50%)

NA

MCP 97% of treatedpatients (complete:90%; partial: 7%) y100% of non-treatedpatients

NA

Anti-FH: anti-complement factor H antibodies; CFH: complementfactor H gene; CFI: complement factor I gene; MCP: membranecofactor protein gene; NA: not available; complete remission:haematological and renal function normalisation; partial remis-sion: haematological normalisation and renal sequels; THBD:thrombomodulin gene.Adapted from Noris et al.12

dialysis, or both, per patient/day), renal function, quality of

usually ineffective except in a few patients with completedeficiency of FH82 (circulating levels of complement proteinsare normal in most patients). Overall, PT is not consideredeffective in patients with isolated MCP mutations, as this isa non-circulating protein attached to the cell membrane, withvirtually all patients relapsing following an episode of aHUSirrespective of the use of PT.12

Even though no prospective clinical trials are available,PT has empirically been the treatment of choice in aHUSfor years as mortality in patients with TTP-HUS decreasedover the last 3 decades. Table 5 summarises the resultsof the largest international registry of PT in patients withaHUS (International Registry of Recurrent and Familial HUS/TTP),including 273 patients diagnosed between 1996 and 2007.12

Complete haematological and renal recovery rates with PT inthis registry are generally below 50% (except for patients withmutations in THBD and MCP), and particularly low rates inpatients with mutations in FH and FI (5 y 12.5%).12 Mortalityand/or outcome of ESRF are generally high in 3 out of 4 patientswith FI mutations. Some papers prove that early intensive PE isessential to prevent patients from developing aHUS, and main-tenance can prevent disease recurrence and ESRF,11,81 thoughthe most effective management strategy is still not known,nor is the long-term impact on renal function.

Concomitant immunosuppression and PT may improveoutcomes in patients with anti-FH antibodies.19,83,84 Highantibody titres are correlated with a higher risk of relapse and

renal sequels in these cases.19 Even though further trials arerequired to conclude on how anti-FH antibodies are devel-oped by patients with aHUS, the fact that the vast majority

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of these patients have complete deficiency of FHR1 leadsto the idea that these antibodies are really targeted againstthe FHR1 protein, and that the anti-FH activity is a crossreaction resulting from the remarkable homology existingbetween these 2 proteins. This possibility is a warning of thepotential anti-FHR1/anti-FH sensitisation in homozygotes forCFHR3-CFHR1 deletion by the exposure to exogenous FHR1,discouraging the use of PI in these individuals.

The potential complications of PI are anaphylactic reac-tions to FFP, hypervolemia, high blood pressure, heart failure,or hyperproteinaemia. The main complications of PE areobstructed venous access (6%), low blood pressure (5%), andallergy (4%)85, with a higher frequency among paediatricpatients.85 A study conducted in 71 paediatric patients withaHUS (59 treated with PE) showed that 80% of children hadsome renal sequels within one month of follow-up, 17%were dialysis-dependant, and 31% developed catheter-relatedcomplications.86

Eculizumab

Eculizumab is a humanised monoclonal IgG2/4 kappa anti-body that binds to the C5 complement protein with highaffinity, blocking the excision into C5a and C5b, and pre-venting the formation of the C5b-9 complex of the terminalcomplement (membrane attack complex) (Fig. 3).4 In aHUS,the dysregulation of the alternative complement pathwayleads to the uncontrolled activation of C5, causing a damageto self structures via the formation of the membrane attackcomplex. This process is rapidly and sustainably reduced asa result of the blockade of the terminal complement pathwayby Eculizumab. A large number of patients with aHUS haveshown a good clinical response to the drug (Table 6).

The efficacy and safety of Eculizumab in aHUS were ini-tially assessed in two phase II, prospective, multicentre trials,including 37 patients older than 12 years of age and withprimary or recurrent disease following RT, who receivedEculizumab for 26 weeks, followed by long-term extensionperiods.5 Seventeen patients with aHUS (mean time fromdiagnosis: 9.7 months) with evidence of progressive TMA fol-lowing ≥4 sessions of PT the week before their inclusion(C08-002) were enrolled in the first study. The second studyrecruited 20 patients (mean time from diagnosis: 48.3 months)receiving PT (1 session every 2 weeks and 3 sessions a week)where no decrease >25% was reported in platelet count forat least 8 weeks prior to first dosing of Eculizumab (C08-003). Genetic or anti-FH antibody mutations were observedin 76% and 70% of patients from the first and the sec-ond study, respectively. Primary outcomes in both studieswere: (a) inhibition of complement-mediated TMA (study 1:increased platelet count; study 2: TMA-free patient ≥12 weeks[no decrease in platelet count >25%, no PT and no dialysis]),and (b) haematological normalisation (≥2 normal consecu-tive measurements of platelets and LDH, with a minimuminterval of 4 weeks). Secondary outcomes included changein the rate of daily interventions for TMA (PI or PE sessions,

life, safety and tolerability. Primary outcomes reported at 26weeks and in extension studies are described in Table 7. Withregard to primary outcome, following 26 weeks of treatment,

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Table 6 – Published cases of patients with atypical haemolytic uraemic syndrome receiving Eculizumab (last updated inApril 2014).

Patients with aHUS in native kidneys

Reference Mutation Response to plasmatherapy

Baseline serumcreatinine, �mol/l

Patient outcome Last serumcreatinine value,

�mol/l

121,122 Unidentified Resistant to plasmaexchange

265 Remission after 3years

35

123 CFH Partially sensitive toplasma exchange

80 Remission after 10weeks

26

124 a Unidentified Resistant to plasmaexchange

690 Recurrence after 2weeks

End-stage renalfailure

125 a Unidentified Resistant to plasmaexchange

∼310 Recurrence after 2weeks

End-stage renalfailure

126 CFH S1191L V1197A Resistant to plasmainfusion

108 Remission after 15months

44

127 CFIp.A258 T

Resistant to plasmaexchange

610 Remission after 7months

230

38,128 b Unidentified No plasma therapy 600 Remission after 6months

125

129 CFHC611Y

Intolerance toplasma exchange

∼230 Remission after 24months

∼100

130 Unidentified Resistant to plasmaexchange

∼325 (dialysis) Remission after 9months

∼80

131 CFH Resistant to plasmaexchange

∼310 (dialysis) Remission after 18months

∼75

132 MCPc.286+ 1G>C

Resistant to plasmaexchange

Dialysis Haematologicalnormalisation

End-stage renalfailure

133 Unidentified Resistant to plasmainfusion

Continuoushemodiafiltration

Remission after oneyear

18

134 CFH3355 G>a;Asp1119Asn; SCR19

Resistant to plasmaexchange

Dialysis Remission > 2.5 years 26

43 a Unidentified Sensitive to plasmaexchange

Dialysis Resolution ofthrombocytopeniaand skin lesions

Dialysis

135 CFH Partially sensitive toplasma exchange

723 (dialysis) Remission Dialysis-free

136 c Unidentified − ∼247 (dialysis) Remission after 6months

Dialysis-free

137 C3 Resistant to plasmatherapy

Dialysis Remission after 2years

Dialysis

138 CFH3514G>T

Resistant to plasmatherapy

∼222 (dialysis) Remission after oneyear

117

139 C3c3466G>A

Resistant to plasmaexchange

Dialysis Remission > 11months

115

140 a C3 – Dialysis Remission Dialysis-free141 c CFB

c.967A>C;p.Lys323Gln

– 20 Favourable outcomeat 6 months, in spiteof the persistence ofslightly increasedplasma levels of LDHy C5b-9

Normal levels

142 CFHp.Arg53Cys; c.157C.T

Resistant to plasmaexchange

Normal levels Remission Normal levels

143 c CFHp.Lys1186ThrCFIp.Ile322Thr

Partially sensitive toplasma exchange

Dialysis Remission 75

144 CFIc.786delA

Resistant to plasmaexchange

Dialysis Remission 88

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Table 6 – (Continued )

Renal transplant patientsPreventive use of Eculizumab

Reference Mutation Previous transplants(number)

Response to plasmatherapy

Baseline serumcreatinine, �mol/l

Patient outcome Last serumcreatinine level,

�mol/l

107 CFH W1183C No Sensitive to plasmaexchange

∼45 No recurrence 44

108 CFH E1198stop No No plasma therapy Dialysis No recurrence Normal109 CFH/CFHR1

hybrid geneNo Sensitive to plasma

exchangeDialysis No recurrence 80

110 CFH/CFHR1hybrid gene

No Sensitive to plasmaexchange

Dialysis No recurrence Normal

111 CFH/CFHR1hybrid gene

No No plasma therapy Dialysis No recurrence 79

112 CFH c.3497C9T No Resistant to plasmaexchange

Dialysis No recurrence 76

Use of Eculizumab for the treatment of post-transplant aHUS recurrence

145 a CFH Y475S Yes (1) Resistant to plasmaexchange

132 Graft loss NS

146,147 C3 R570Q Yes (1) Sensitive to plasmaexchange

320 2 recurrences incasesof delayedEculizumab

230

97 Unspecified No Resistant to plasmaexchange

323 Remission 238

98 CFH S1191L Yes (2) Intolerance toplasma exchange

131 Remission 130

148 a Unidentified No Resistant to plasmaexchange

415 Graft loss NS

42 CFH Yes (1) Resistant to plasmaexchange

500 Remission 62

69 C3 R570W Yes (2) Partially sensitive toplasma exchange

220 Remission 115

99 CFHE3514Stop

No Partially sensitive toplasma exchange

565 (dialysis) Remission 229

149 Unidentified Yes (1) Resistant to plasmaexchange

449 (dialysis) Recurrence 5months followingwithdrawal ofEculizumab. Graftloss

NS

43 CFH No Partially sensitive toplasma infusiond

220 Remission(disappearance ofskin lesions)

209

aHUS: atypical haemolytic uraemic syndrome; CFB: complement factor B gene; CFH/CFHR1: complement hybrid gene resulting from CFH/CFHR1conversion; CFH: complement factor H gene; CFHR1: complement factor H-related protein 1 gene; CFI: complement factor I gene; HUS: haemolyticuraemic syndrome; LDH: lactate dehydrogenase; TMA: thrombotic microangiopathy; NE: not specified.a Receiving only one dose of Eculizumab.b Reduced dose of Eculizumab.c Early use of Eculizumab (≤7 days following diagnosis of aHUS).d Suspected persistence of TMA activity due to the presence of ulcerative skin lesions in lower limbs.

in study 1 treatment with Eculizumab was associated witha significant increase in the number of platelets from base-line (p < 0.001) and a rate of haematological normalisation of76%. In study 2, 80% of patients were free of TMA episodes

following 26 weeks of treatment with Eculizumab and 90%had haematological normalisation. In terms of secondary out-comes, treatment with Eculizumab at 26 weeks was associatedwith a significant reduction in the rate of daily interventions

for TMA vs. baseline (p < 0.001), as well with a continuousimprovement of estimated GFR (+32 ml/min/1.73 m2 [p = 0.001vs. baseline] and +6 ml/min/1.73 m2 [p < 0.001 vs. baseline]in studies 1 and 2, respectively), a decrease in proteinuria

(p < 0.05) and a reduced need for dialysis. Also, the earlierEculizumab is introduced in trials (less time of evolutionbetween the clinical manifestation of aHUS and the drug),the more significant the improvement of the estimated GFR

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Table 7 – Main results from prospective studies with Eculizumab in patients with aHUS.

C08-002 (n = 17) C08-003 (n = 20) C10-003 (n = 22) C10-004 (N = 41)

Week 26 Week 64 Week 100 Week 26 Week 62 Week 156 Week 26 Week 26

Change in platelet count from baseline (×109/l), meanp value vs. baseline

+73a

<0.001+910.001

+97<0.0001

+5NS

NA NA +164<0.0001

+135<0.0001

Platelet count normalisation,b number of patients (%) 14 (82) 15 (88) NA NA NA NA 21 (95) 40 (98)No TMA events,c number of patients (%) 15 (88) 15 (88) 15 (88) 16 (80)a 17 (85) 19 (95) 21 (95) 37 (90)Haematological normalisation (complete

haematological response)d, number of patients (%)13 (76) 15 (88) 15 (88) 18 (90) 18 (90) 18 (90) 18 (82) 36 (88)

Daily intervention rate for TMAe (number ofevents/patients/day)Before Eculizumab, meanOn Eculizumab, meanp value vs. “before Eculizumab”

0.880<0.001

0.880<0.001

NA 0.230<0.001

0.230<0.001

NA NA NA

Complete response for TMA,f number of patients (%) 11 (65) 13 (76) NA 5 (25) 7 (35) NA 14 (64)a 30 (73)a

Outcome of estimated GFR (ml/min/1.73 m2)p value vs. baseline

+320.001

+32<0.001

+38≤0.05

+6<0.001

+90.003

+4NS

+64<0.0001

+29<0.0001

Reduction ≥ 25% in serum creatinine, number ofpatients (%)

11 (65) 13 (76) 13 (76) 3 (15) 7 (35) 11 (55) 16 (73) NA

Increased estimated GFR ≥ 15 ml/min/1.73 m2, numberof patients (%)

8 (47) 9 (53) 10 (59) 1 (5) 3 (15) 8 (40) 19 (86) 22 (54)

No dialysis, number of patients/number of patients indialysis at the start of treatment (%)

4/5 (80) 4/5 (80) NA 0/2 (0) 0/2 (0) NA 9/11 (82) 20/24 (83)

Improvement in CRD in at least one stage, number ofpatients (%)

10 (59) 11 (65) 13 (76) 7 (35) 9 (45) 12 (60) 17 (77) 26 (63)

Reduction of proteinuria in at least one grade inpatients with baseline proteinuria grade ≥ 1, numberof patients/total number of patients

12/15 9/11 NA 6/11 7/9 NA NA NA

Improvement in quality of life (change in questionnairescoring), meang

p value vs. baseline

+0.32<0.001

+0.30<0.001

+0.29<0.0001

+0.10<0.001

+0.13<0.001

+0.16≤0.001

+19.7<0.0001

+0.230.003

aHUS: haemolytic uraemic syndrome; CRD: chronic renal disease; EQ-5D: EuroQoL Group 5-Dimension Self-Report Questionnaire; FACIT-F: Functional assessment of chronic illness therapy-fatigue;GFR: glomerular filtration rate; LDH: lactate dehydrogenase; NA: not available; NS: not significant; PE: plasma exchange; PI: plasma infusion; TMA: thrombotic microangiopathy.a Primary outcome.b Platelets ≥150 × 109/l.c No platelet decrease > 25%; no plasma therapy and no start of dialysis for ≥12 weeks.d ≥2 normal consecutive measurements of platelets and LDH, with at least 4 weeks apart.e Plasma PI or PE sessions, dialysis, or both, per patient/day.f Complete haematological response and decrease ≥25% in serum creatinine from baseline (two consecutive measurements with at least 4 weeks apart).g The EQ-5D questionnaire was used in all studies, except for C10-003 study, where the FACIT-F questionnaire for paediatric patients was used.

Adapted from Legendre et al.,5 Delmas et al.,87 Gaber et al.,88 Fakhouri et al.90 and Greenbaum et al.91

0 1 5

aHUS and a continuous risk of TMA and potential organ92

436 n e f r o l o g i a. 2

(p < 0.05). The results of extension trials at 1 and 3 yearsprove that long-term treatment with Eculizumab is associ-ated with maintenance or progressive improvement of thehaematological response and renal function.87,88 All thesepositive results with Eculizumab were observed irrespec-tively in patients with or without genetic or anti-FH antibodyabnormalities.

Overall, tolerability of Eculizumab was good, and severetreatment-related adverse effects were only reported in 4patients in each study, probably in the context of the back-ground condition, aHUS (study 1: malignant hypertension,2

severe hypertension, and asymptomatic bacteriuria; study2: influenza infection, peritonitis, venous sclerosis at thetime of the infusion, and fever Q). Increased risk of infec-tion by encapsulated germs, especially Neisseria meningitides,results from the mode of action of Eculizumab, and soall patients were vaccinated against Neisseria (tetravalentvaccine) 14 days before treatment and/or received antibi-otics, with no reports of meningitis. Only one patientdied of digestive bleeding 3 years later (unrelated toEculizumab).

The safety and efficacy of Eculizumab during pregnancyhas recently been studied in patients with paroxysmalnocturnal haemoglobinuria. Data from 61 women with 75pregnancies while on treatment with Eculizumab suggest agood tolerability profile of Eculizumab during pregnancy, withhigh rates of foetal and maternal survival (96% and 100%,respectively). Eculizumab was observed in 7 out of 20 bloodsamples of umbilical cord and in none of the 10 samples ofhuman milk.89

Two phase III, multicentre, prospective, open-label trialsof Eculizumab in aHUS are currently ongoing, one in adultpatients (n = 41; C10-004)90 and the other in paediatric patients

91

(n = 22; C10-003 22). Unlike previous trials, these new studiesprimarily involve patients who have been recently diagnosedwith aHUS (73%). In the adult trial, mean time from diagnosis

Suspicion based on aHUS

Monitor the treatment

Paediatric patient: Start eculizumab earlier as treatment of cho

±Support treatment

Adult patient*: Start eculizumab earlier (perform early and intense

starting it, if it is this that could delay it) ±

Support treatment

Fig. 5 – Treatment for atypical haemolytic uraemic syndrome. PEsyndrome. * When the diagnosis of aHUS is unequivocal (positivrenal transplant), Eculizumab should be use earlier as treatment

;3 5(5):421–447

to inclusion was ∼3 weeks, whereas mean time from clinicalonset to Eculizumab was 2 weeks (15% of patients had notbeen treated with PT prior to Eculizumab).90 The paediatrictrial involves patients with aHUS ≤18 years with a mean timefrom diagnosis to inclusion of ∼2 weeks and a mean timefrom clinical onset to Eculizumab of ∼1 week (55% had notbeen treated with PT).91 Results from these 26-week trialsconfirm the significant haematological and renal functionimprovements seen in previous studies, as well as the benefitsof early use of Eculizumab (Table 7).90,91 The safety profilewas similar, although two patients developed meningococcalmeningitis (5%) in the adult study. Both infections werewell-managed, and one of the patients continued treatmentwith Eculizumab. Survival in patients from both studieswas 100%.

Blood samples from adult patients enrolled in the C10-004 trial were collected at baseline (before treatment withEculizumab) and in subsequent visits up to Weeks 49–54in order to measure the impact of Eculizumab on biomark-ers related to TMA and endothelial damage in aHUS.92

Patients with aHUS (irrespective of the presence or absenceof related mutations, PT, or haematological values) showedactivated complement system and inflammation, coagula-tion, activation, endothelial damage, and renal injuries priorto treatment. Eculizumab led to normalisation of comple-ment activation-related biomarkers and significantly reducedbiomarkers of inflammation, thrombotic risk, endothelialand organic damage. Eculizumab also reduced biomark-ers related to the activation of the alternative complementpathway and endothelial activation, although they did notcompletely return to normal. These findings underline thepersistent chronic of complement activation in patients with

damage.There is also a retrospective study including 19 paediatric

patients with aHUS receiving Eculizumab in a clinical setting

• Draw blood specimens before starting the treatment for subsequent studies. • Meningococcal vaccination ± Antibiotic prophylaxis

ice

PEs until

: plasma exchange; aHUS: atypical haemolytic uraemice personal or family history or disease recurrence following

of choice.

1 5;3 5

fo6wwftnra

Ra

Eoapitbdb

T

I(tttpetslouhtrcPtodri

tsmbaotiss

n e f r o l o g i a. 2 0

or a mean time of 28 weeks (C09-001).3 Eighty nine percentf patients in this study had a normalised platelet count and8% remained free of TMA episodes, although most of themere under haematological remission with prior treatmentith PE. The rate of interventions for TMA was reduced

rom 0.3 per patient/week to 0 (p < 0.0001). GFR increasedo ≥15 ml/min/1.73 m2 in 47% of patients and 50% requiredo dialysis at all. Pyrexia (47%), diarrhoea (32%), and upperespiratory tract infections (32%) were the most commondverse effects.

ecommendations for the management of thetypical haemolytic uraemic syndrome

ver since Eculizumab was approved for the treatmentf aHUS by the EMA and the Spanish Agency for Drugsnd Health Products (AEMPS) in 2011,3 management andrognosis among patients with aHUS have substantially

mproved, as the drug was approved for use as first-lineherapy. Recommendations for the treatment of aHUS madey the authors of this document based on available evi-ence and cumulative clinical experience are summarisedelow.

reatment of atypical haemolytic uraemic syndrome

n view of technical difficulties of PT in paediatric patientsby body size) and potential complications, in addition tohe superiority of Eculizumab for the recovery of renal func-ion (and the resulting improved prognosis), early first-linereatment with Eculizumab is highly recommended in thisopulation, therefore avoiding the use of PE. Consequently,arly administration of Eculizumab is recommended asreatment of choice in first line in paediatric patients withuspected aHUS (Fig. 5).91 Eculizumab should be initiated ear-ier in adult patients with suspected aHUS following PE.90 PE isnly recommended in adults when diagnosis is unclear. These of Eculizumab may only be disregarded in patients withaematological complete recovery and improved renal func-

ion following PE. In this study, the aHUS French Study Groupecommended switching the patient to Eculizumab if plateletount or LDH levels do not return to normal following the fifthE, or reducing plasma creatinine ≥25%.93 Eculizumab is thereatment of choice recommended whenever the diagnosisf aHUS is unequivocal (positive personal or family history orisease recurrence following RT). Early administration ensureseversibility of haematological parameters and prevents renalnjuries.

All patients should be vaccinated against N. meningi-ides (preferably with conjugate tetravalent vaccines againsterotypes A, C, Y, and W135, and serotype B) prior to treat-ent with Eculizumab. If treatment with Eculizumab cannot

e delayed until vaccine response, associated treatment withntibiotics against N. meningitides may be initiated and antibi-tic prophylaxis may be established3 as per the hospital pro-

ocol. Given the higher frequency of invasive meningococcalnfection among paediatric patients, and the absence oferotype B protection (currently the most prevalent followingystemic vaccination in the population for other serotypes),

(5):421–447 437

this age group should continue to use antibiotic prophy-laxis, including penicillin or amoxicillin, in combination withEculizumab,2 although these prophylaxis protocols may beadjusted following the recent availability of the new serotypeB vaccine. The maintenance of antibiotic prophylaxis in adultpatients receiving Eculizumab is at the physician’s own discre-tion and should be individually assessed. Continued antibioticprophylaxis should be considered in immunodepressedpatients receiving Eculizumab as a result of the lower responseto the vaccine among these patients (particularly amongthose receiving renal transplants). In paediatric patients,Haemophilus influenzae and pneumococcal vaccines are alsorequired, together with strong compliance of local effec-tive recommendations on compulsory vaccines for each agegroup.

If response to Eculizumab is good, treatment should bemaintained indefinitely as recommended in the summary ofproduct characteristics.3 No recommendations can be madeso far on the right treatment duration, though increasing expe-rience with drug use may help better define this as well astreatment strategies in the future.

Withdrawal and/or individual dose titration of Eculizumabmay be considered for specific cases, but only among low-riskpatients (with isolated mutation in MCP and negative familyhistory), always individually and following at least 12 monthsof treatment.94,95 Patients who withdraw treatment as perclinical indication should be carefully monitored for at least12 weeks for potential abnormalities suggestive of TMA and/orrelapse.3,96 In these patients immediate readministration withEculizumab should be considered.3,96

Eculizumab should be maintained for at least 3 months inpatients with aHUS and acute renal failure requiring dialysisin order to assess the improvement of renal function. Pro-gressive increase of diuresis with good pressure managementare positive parameters guiding the management of the TMAprocess and improvement of renal injuries. Renal biopsiesin patients receiving dialysis may help decision-makingassociated with treatment continuation. If treatment failsin patients and they still require dialysis for renal failure,Eculizumab should be withdrawn, except in patients with sys-temic disease manifestations, where treatment continuationshould be assessed individually.

Whenever PT is considered for a patient with aHUS,PE should preferably be performed with FFP replacement(1.5 per plasma volume [60–75 ml/kg] per session to addcomplement factors). Sessions should be performed untilplatelet count returns to normal, end of haemolysis ends,and sustained improvement of renal function for severaldays occurs. Five weekly sessions should be conducted there-after for the first 2 weeks and 3 weekly sessions for thenext 2 weeks, while PE continuation should be individuallyassessed.2,82

Patients with aHUS who develop anti-FH antibodies whileon PT should use concomitant immunosuppressants to pre-vent antibody formation.19,73,83,84 Treatment response amongthese patients should be monitored based on the outcome ofantibody titres.73

General supportive measures are necessary to ensureacceptable conditions for patients until TMA is managed.High blood pressure is common among patients with aHUS

438 n e f r o l o g i a. 2 0 1 5;3 5(5):421–447

Table 8 – Differential diagnosis of TMA in renal transplant patients.

TMA de novo Recurrence of aHUS

History of HUS/TMA No YesSystemic involvement No CommonIntensity of clinical picture Mild SevereOnset Progressive SuddenIntensity of haematological TMA Low HighCausative/triggering agents CNI, mTORiViral infectionsHumoral rejection Triggering factors are not always foundReversibility Yes No. Graft loss

aHUS: atypical haemolytic uraemic syndrome; CNI: calcineurin inhibitors; HUS: haemolytic uraemic syndrome; mTORi: mammalian target ofRapamycin inhibitors; TMA: thrombotic microangiopathy.Adapted from Zuber et al.68

and should be treated with angiotensin II blockers (ACEIor AIIRA). Volemic control is also vital due to commonhypervolemia and acute risk of pulmonary oedema. Trans-fusions of red blood cells concentrates and/or the use oferythropoiesis-stimulating factors should be considered forthe treatment of anaemia. Platelet transfusions should bereserved for cases of severe low platelet counts (<30,000/mm3)or exceptionally for severe bleeding and/or prior to invasiveprocedures with risk of bleeding, as the TMA phenomenonmay become worse. Potential agents leading to aHUS shouldalso be identified and managed. Paediatric patients withaHUS should be referred to specialised centres for paedi-atric nephrology including expert staff and an intensive careunit for paediatric patients in order to ensure adequatetreatment.

Atypical haemolytic uraemic syndrome andtransplantation

Treatment for the recurrence of atypical haemolytic uraemicsyndrome in renal transplantationPrimary clinical criteria for the differential diagnosis ofthe different types of TMA in RT and an algorithm for themanagement of these entities are described in Table 8and Fig. 6, respectively. Diagnosis in patients receivingrenal transplantation and developing TMA, with a his-tory of aHUS episodes before transplantation, should bedirected to disease recurrence (ruling out other potentialcauses).

Treatment for the recurrence of aHUS in patients receiv-ing renal transplants should be performed under thesame terms as in aHUS of native kidneys by means ofthe early use of Eculizumab.5,42,43,69,90,91,97,100 In view ofthe induced immunosuppression status of transplantedpatients (chronic immunosuppression), vaccination againstN. meningitides is also recommended to assess the main-tenance of antibiotic prophylaxis while on treatment withEculizumab.

In patients with progressive TMA in the first study of

Eculizumab, renal function recovery following administrationwas significantly improved in the long term in nontrans-planted patients (with aHUS in native kidneys) comparedto patients receiving renal transplantation.5 This result may

be related to the fact that transplanted patients enrolled inthe study received Eculizumab later than nontransplantedpatients (mean time from clinical onset to enrolment: 1.71and 0.67 months for both types of patients, respectively). Thisobservation stresses the need to early use Eculizumab foraHUS recurrence in RT.

Prophylaxis for the recurrence of the atypical haemolyticuraemic syndrome following renal transplantationPerspectives of RT in patients with ESRF secondary to aHUSreceiving dialysis have significantly changed in recent years,especially in patients with high risk of disease recurrencefollowing transplantation (patients with risk mutation and/orrelapse of aHUS). The use of Eculizumab has made treatmentoptions available for these patients for whom RT was con-traindicated because of the high rate of relapse and the risk ofrenal graft loss. There are currently three treatment optionsto prevent aHUS recurrence following RT: (a) a liver–kidneycombined transplantation; (b) simple RT together withprophylaxis with PT, and (c) simple RT with prophylacticEculizumab.

Over 20 cases of patients with aHUS and mutations ingenes coding for complement factors primarily synthesisedin the liver (FH, FB or FI) and receiving liver transplants(isolated or combined with RT) have been recently reportedso as to avoid the consequences of the genetic defect andprevent disease recurrence. This strategy, combined withthe perisurgical use of Eculizumab or plasma (to eliminatedysfunctional complement factors during surgery and addenough functional factors until liver function is recovered),was successful in several occasions, with good liver func-tion and no recurrence of aHUS during follow-up.70,101–105

Nonetheless, given the potential morbi-mortality related toliver transplants,2 and the challenge posed by the availabil-ity of organs and safer alternatives, liver–kidney transplantsshould only be considered in selected patients with aHUS asa second option. Isolated liver transplants are also not rec-ommended for patients with aHUS and functional kidneys, aschronic immunosuppression risks outweigh risks related to

68

long-term use of Eculizumab.Prophylaxis with PT in patients with ESRF secondary to

aHUS and receiving simple RT has been associated with pos-itive outcomes in terms of disease recurrence prevention in

1 5;3 5

sfsioia

wptawrpTvifCtwtE8l7

Fimht

n e f r o l o g i a. 2 0

everal papers.68 Yet, recurrence of aHUS has been reportedollowing RT in patients at risk, including the use of inten-ive PT.68,100 On the other hand, factors including the potentialncreased risk of disease recurrence with progressive spacingf PT sessions106 or the impact on QoL in patients result-

ng from long-term PT (mostly related to sustained vascularccess) are restrictive of this strategy.68

Several positive experiences with the use of prophylaxisith Eculizumab among paediatric patients receiving FHrior to RT from a cadaveric donor have been reported inhe last years (Table 6),107–111 therefore suggesting that RTssociated with prophylactic Eculizumab is an effective andell-tolerated option for these patients.68 Zuber et al.100 have

ecently published a series including 9 patients receiving pro-hylaxis with Eculizumab for aHUS recurrence following RT.100

his series involves 6 paediatric patients and 3 adults witharious mutations in the alternative complement pathway (5n FH, one in C3 and 3 patients with hybrid genes resultingrom the non-homologous recombination between CFH andFHR1). Two patients receiving post-RT PE were later switchedo Eculizumab, two patients received Eculizumab from theeek before transplantation (unrelated living donor; urgent

ransplant from cadaveric donor) and the remaining 5 receivedculizumab immediately following transplantation. The other

patients had a positive outcome without recurrence fol-owing a mean follow-up of 14.5 months (mean creatinine:1.6 ± 44.8 �mol/l), except for one case of early thrombosis

AHR

TMA in a renal transplanted patienanaemia + thrombopenia +

De novo

Viral infection(CMV, virus BK)

Drugs(ICN, imTOR)

Aetiological treatment± PEs

Modification IS± PEs

Evaluate Eculizumabin resistant cases

ig. 6 – Treatment for thrombotic microangiopathy in renal transnhibitors; mTORi: mTOR inhibitors (mammalian target of Rapam

icroangiopathy; AHR: acute humoral rejection; PE: plasma exchaemolytic uraemic syndrome; STEC: Shiga toxin-producing Esch

ransplantation is very rare.

(5):421–447 439

leading to graft loss. Blasco Pelicano et al.112 reported the firstuse of prophylactic Eculizumab in our country in an adultfemale patient with FH mutation and a favourable outcome,without signs of relapse following 3 years of RT.112 Patientswith ESRF secondary to aHUS who are eligible for RT shouldtherefore use prophylactic Eculizumab as a first option for theprevention of aHUS recurrence following transplantation.68,93

Fig. 7 includes an algorithm for the previous assess-ment and management of aHUS patients who are eligiblefor RT.

There are also positive experiences on liver–kidneytransplants using prophylactic Eculizumab,105 although asexplained before, liver–kidney transplants should be consid-ered as a second option for selected patients with aHUS.

Living donor transplants have traditionally been con-traindicated in patients with aHUS in view of the high rates ofdisease recurrence and graft loss, and the risk of undetectablemutations in the complement system of the donor poten-tially resulting in the subsequent development of aHUS.1,66,113

Progress made so far in the field of genetic diagnosis and theavailability of Eculizumab allow for consideration of relatedliving donor transplantation as a valid option for patients withaHUS. A complete genetic-molecular exam should alwaysbe performed in donors, who will only be eligible based on

mutations identified in the patient and absent in the donor.However, if the related donor and the recipient share anygenetic factor of susceptibility to aHUS, or if no mutations

Aetiological treatment± PEs

aHUS

Recurrence

t (non-immune haemolytic graft dysfunction)

STEC-aHUS

Eculizumabearlier

plantation. CMV: cytomegalovirus; CNI: calcineurinycin); IS: immunosuppression; TMA: thromboticange; HUS: haemolytic uraemic syndrome; aHUS: atypicalerichia coli. a STEC-HUS recurrence following renal

440 n e f r o l o g i a. 2 0 1 5;3 5(5):421–447

Low risk of aHUS recurrence after KT

Patient with CTKD secondary to aHUScandidate for KT

Moderate-high risk of aHUS recurrence after KT

- Mutations in CFH, CFI C3, CFB- CFH/CFHR1 Hybrid gene Combined mutations- Mutation unidentified / with unknown effect - Persistence of anti-FH antibodies - Previous recurrence of aHUS (in an affected individual or family)- With no mutation but with CFH polymorphisms

- Isolated mutations in MCP- DGKE mutation- Undetectable-FH antibodies

• Complement study.a • DGKE study if the HUS was produced in the first year of life

Cadaver donor With unrelated

live donor

Cadaver donor With unrelated

live donor.b

Related livedonor

Related livedonor

Unable to perform KT with related

living donor

Unable to performKT with related

live donor

Yes

KT with no prophylaxis

No Yes No

KT with eculizumab prophylaxisas first option

The mutation indisputablyassociated with the pathogenesis

of an aHUS in the receptornot found in the donor

The mutation indisputably associated with the pathogenesis of an aHUS in the receptor not found in the donor

Eculizumab is available

Fig. 7 – Recommendations for the management of patients with ESRF secondary to aHUS who are eligible for RT. Adaptedfrom Zuber et al.68 aHUS: atypical haemolytic uraemic syndrome; CFB: complement factor B gene; CFH/CFHR1: complementhybrid gene resulting from CFH/CFHR1 conversion; CFH: complement factor H gene; CFHR1: complement factor H relatedprotein 1; CFI: complement factor I gene; DGKE: Diacylglycerol Kinase, Epsilon 64 kDa; ESRF: end-stage chronic renal failure;FH: complement factor H; MCP: membrane cofactor protein gene; RT: renal transplantation. CKTD: chronic terminal kidneydisease; KT: kidney transplant. a Determination of plasma levels of C3, C4, FH, FI and FB, as well as MCP expression inperipheral leukocytes; complete genetic study to detect known complement mutations (and risk polymorphisms), togetherwith screening for anti-FH antibodies. b RT with an unrelated living donor will only be considered if Eculizumab is available.

are observed both in the recipient or the donor, no geneticallymatched living donor transplantation should be performed.93

Also, a living donor transplantation should only be consideredif Eculizumab is available.

No specific protocols based on prospective trial onimmunosuppression to reduce the risk of post-RT relapseare available. In general, calcineurin inhibitors114 and mTORinhibitors68,115 are thought to be related to post-RT TMA, witha synergic effect in the combination of both drugs.116 Thesedrugs should therefore be carefully used in patients receivingrenal transplants due to ESRF secondary to aHUS. Guidelineson immunosuppression based on belatacept could be fol-lowed depending on the immunological risk of each patient,

although no conclusive data are available in the literature as tothe best immunosuppressive strategy for the risk population.

Conclusions

• aHUS results from genetic or acquired dysregulation of theactivated alternative complement pathway on cell surfacesleading to systemic TMA. Several mutations and polymor-phisms have been described in recent years in genes fromcertain complement factors associated with this dysregula-tion.

• Clinical presentation comprises the triad of nonimmunemicroangiopathic haemolytic anaemia, thrombocytopenia,and acute renal dysfunction, associated with commonextra-renal manifestations. Before Eculizumab became

available, aHUS was generally associated with increasedmortality and/or progression to ESRF, as well as to increasedrecurrence following RT.

1 5;3 5

•

•

•

•

•

A

TSt

S

Rf(tÁ

fSww

C

DdpPcDitap

Although unnecessary to the clinical diagnosis of aHUS, aresearch of the complement including plasma levels of all

Table 9 – Protocol for sample collection for complementtests in patients with atypical haemolytic uraemicsyndrome.

• If samples are to be sent to a reference laboratory, westrongly recommend to contact the laboratory before andfollow the instructions on samples required for assays

• If no reference laboratory is available and samples are to bestored for further tests, samples to be collected are:• 10 ml of blood with EDTA• 10 ml of coagulated blood for serum

• If a patient is a young child and 20 ml of blood cannot bedrawn, 2–3 ml can be collected from each of the samples(6–9 ml in total)

• Blood with EDTA must be immediately centrifuged at3000 rpm for 10 min at 4 ◦C followed by plasma collection ina clean tube, taking care so as not to absorb any red cells.After labelling 5 tubes with the patient’s name, the date, andan indication showing EDTA plasma, plasma collected fromthese tubes should be distributed, freezing themimmediately and keeping them at a temperature of −80 ◦C

• Once plasma was drawn, store the cellular pellet frozen at−80 ◦C for future DNA collection

• Coagulated blood for 1 h at room temperature should becentrifuged at 3000 rpm for 10 min at 4 ◦C and the serumshould be immediately separated, taking care so as not toabsorb any red cells in the clean tube. Following labelling of5 tubes with the patient’s name, the date, and an indicationshowing serum, serum collected from these tubes should be

n e f r o l o g i a. 2 0

Diagnosis should point to aHUS if TMA is clinically sus-pected and provided that the Shiga toxin/STEC test isnegative, plasma ADAMTS13 is >5–10% and if secondaryforms of HUS have been ruled out.

Eculizumab is a monoclonal antibody inhibiting C5 acti-vation and the formation of the membrane attackcomplex, responsible for damage to self structures inaHUS. In prospective studies including patients with aHUS,Eculizumab effectively prevented the TMA process and wasassociated with long-term significant haematological andrenal function improvements.

The authors of this paper recommend the early use ofEculizumab for aHUS in paediatric and adult patients withclinically suspected aHUS in native kidneys, and aHUSrecurrence following RT or post-RT de novo aHUS.

Prophylaxis with Eculizumab is recommended for the pre-vention of aHUS recurrence in patients with secondary ESRFreceiving RT from a living or cadaveric donor.

Eculizumab should be considered in patients with sec-ondary TMA refractory to usual treatment.

pproval

his document has been approved by the Spanish Transplantociety (SET), the Spanish Society of Nephrology (SEN), andhe Spanish Association of Paediatric Nephrology (AENP).

ources of funding and acknowledgements

esearch carried out by Dr. Rodríguez de Córdoba has beenunded by the Ministry of Economy and CompetitivenessSAF2011-26583), the Community of Madrid (S2010/BMD2316),he European Union (EURenOmics), the Fundación Renal Inigolvarez de Toledo, and the CIBER of rare diseases.

The authors would like to thank Alexion Pharmaceuticalsor the logistical support provided for meetings held by thepanish Group for atypical haemolytic uraemic syndrome, asell as for the editorial assistance provided by Ogilvy Health-orld.

onflicts of interest

r. Ariceta, Dr. Blasco, Dr. Campistol, Dr. Praga, Dr. Rodrígueze Córdoba, Dr. Valdés, Dr. Vilalta and Dr. Román haverovided consultancy and teaching assistance to Alexionharmaceuticals. Dr. Torra has been a member of the expertommittees in aHUS sponsored by Alexion Pharmaceuticals.r. Espinosa has taken part in clinical trials sponsored by Alex-

on Pharmaceuticals. None of these activities have influencedhe development and interpretation of this manuscript, theuthors being solely responsible for the contents herein dis-layed.

(5):421–447 441

Annex 1. Recommendations for the treatmentof secondary forms of haemolytic uraemicsyndrome

The management of secondary forms of HUS should be basedon the treatment of the primary aetiology of the disease andthe administration of PE. As explained, there is an increas-ing number of reports in the literature from patients withsecondary HUS showing good response to Eculizumab, there-fore suggesting that the complement plays an importantrole in the development of TMA in certain patients withsecondary HUS.30–34,117–120 Consequently, the potentially tem-poral administration of Eculizumab should be considered assalvage therapy in selected patients with secondary HUSrefractory to initial treatment measures. Also, screening forcomplement gene mutations in patients with secondary HUSpoints out that the underlying disease is actually aHUS, andso these patients should be treated as per the guidelinesdescribed in “Recommendations for the management of theatypical haemolytic uraemic syndrome”.

Annex 2. Recommendations of interest for thediagnosis of genetic abnormalities

distributed, freezing samples immediately after andmaintaining them at −80 ◦C

EDTA: ethylenediaminetetraacetic acid.

0 1 5

r

442 n e f r o l o g i a. 2

factors should be conducted, together with a complete geneticanalysis of all affected patients. Samples should be collectedprior to treatment initiation, including plasma exchange,and should be sent to a reference laboratory (Table 9). Asmutations have been identified in the complement sys-tem of patients with secondary STEC-HUS and HUS, genetictests should also be individually conducted in these patientsto study the potential association with complement/aHUSabnormalities.2

Genetic diagnosis of complement genes is generally recom-mended as it allows for individualised estimates of prognosisand risk of disease recurrence. Genetic tests are essential forpatients who may be eligible for RT.

A Working Group coordinating and providing online coun-selling on complement tests for aHUS patients has been setup in order to collect data and conduct further trials.

Graft samples from renal transplant patients with aHUSshould be collected for future studies.

Annex 3. Recommendations for thedetermination of ADAMTS13

Table 10.

Table 10 – Protocol for collection and shipping ofsamples for determination of ADAMTS13 activity.

• Collect blood by venopuncture in two 5 ml citrate tubes• Label tubes with patient’s name and last name, date, and

time of collection• Centrifuge tubes for 7 min at 3500 rpm• Transfer supernatant (plasma) to Eppendorf’s tubes for

aliquots. Avoid transferring part of the precipitate (resultsmay be altered). Samples must not be frozen before this step

• Label new tubes with the same information• Freeze tubes at −20 ◦C and send them to a reference

laboratory. Also send a 5 ml EDTA tube (refrigerated)

ADAMTS13: A Disintegrin And Metalloproteinase with a Throm-boSpondin type 1 motif, member 13; EDTA: ethylenediaminetetra-acetic acid.

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3. Alexion Pharmaceuticals I. Soliris (eculizumab). Fichatécnica; 2012.

4. Rother RP, Rollins SA, Mojcik CF, Brodsky RA, Bell L.Discovery and development of the complement inhibitoreculizumab for the treatment of paroxysmal nocturnalhemoglobinuria. Nat Biotechnol. 2007;25:1256–64.

5. Legendre CM, Licht C, Muus P, Greenbaum LA, Babu S,

Bedrosian C, et al. Terminal complement inhibitoreculizumab in atypical hemolytic-uremic syndrome. NEngl J Med. 2013;368:2169–81.

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6. Campistol JM, Arias M, Ariceta G, Blasco M, Espinosa M,Grinyo JM, et al. An update for atypical haemolyticuraemic syndrome: diagnosis and treatment. Aconsensus document. Nefrologia. 2013;33:27–45.

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9. Siegler R, Oakes R. Hemolytic uremic syndrome:pathogenesis, treatment, and outcome. Curr OpinPediatr. 2005;17:200–4.

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